Carboxymethyl cellulose from thermotoga maritima

ABSTRACT

A purified thermostable enzyme is derived from the eubacterium  T. maritima . The enzyme has a molecular weight as determined by gel electrophoresis of about 35 kilodaltons and has cellulose activity. The enzyme can be produced from native or recombinant host cells and can be used to aid in the digestion of cellulose where desired.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.08/951,889, filed on Oct. 16, 1997, issued as U.S. Pat. No. 6,008,032 onDec. 28, 1999; which is a divisional of prior U.S. application Ser. No.08/518,615, filed Aug. 23, 1995, issued as U.S. Pat. No. 5,962,258 onOct. 5, 1999, the contents of which are incorporated by reference intheir entirety herein.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production and isolation of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention has been putatively identified as an endoglucanaseand in particular an enzyme having carboxymethyl cellulose activity,sometimes hereinafter referred to as “CMCase”.

Cellulose, a fibrous, tough, water-insoluble substance is found in thecell walls of plants, particularly, in stalks, stems, trunks and all thewoody portions of plant tissues. Cellulose constitutes much of the massof wood, and cotton is almost pure cellulose. Because cellulose is alinear, unbranched homopolysaccharide of 10,000 to 15,000 D-glucoseunits, it resembles amylose and the main chains of glycogen. But thereis a very important difference; in cellulose, the glucose residues havethe beta configuration, whereas in amylose, amylopectin and glycogen theglucose is in the alpha configuration. The glucose residues in celluloseare linked by (beta 1→4) glycosidic bonds. This difference givescellulose and amylose very different 3-dimensional structures andphysical properties.

Cellulose cannot be used by most animals as a source of stored fuel,because the (beta 1→4) linkages of cellulose are not hydrolyzed byalpha-amylases. Termites readily digest cellulose but only because theirintestinal tract harbors a symbiotic microorganism, trichonympha, whichsecretes cellulose, an enzyme that hydrolyzes (beta 1→4) linkagesbetween glucose units. The only vertebrates able to use cellulose asfood are cattle and other ruminant animals (sheep, goats, camels andgiraffes). The extra stomachs “rumens” of these animals teem withbacteria and protists that secrete cellulose.

The enzymatic hydrolysis of cellulose is considered to require theaction of both endoglucanases (1,4-beta-D-glucan glucanohydrolase) andexoglucanases (1,4-beta-D-glucan cellobiohydrolase). A synergisticinteraction of these enzymes is necessary for the complete hydrolysis ofcrystalline cellulose. (Caughlin, M. P., Genet. Eng. Rev., 3:39-109(1985). For the complete degradation of cellulose (cellulose toglucose), β-glucosidase might be required if the “exo” enzyme does notrelease glucose. 1,4-β-d-Glucan glucohydrolase is another type of “exo”cellulose.

Thermophilic bacteria have received considerable attention as sources ofhighly active and thermostable cellulolytic and xylanlytic enzymes(Bronneomeier, K. and Staudenbauer, W. L., D. R. Woods (Ed.), TheClostridia and Biotechnology, Butterworth Publishers, Stoneham, Mass.(1993). Recently, the most extremely thermophilic organotrophiceubacteria presently known have been isolated and characterized. Thesebacteria, which belong to the genus Thermotoga, are fermentativemicroorganisms metabolizing a variety of carbohydrates (Huber, R. andStetter, K. O., in Ballows, et al., (Ed.), the procaryotes, 2nd Ed.,Springer-Verlaz, New York, pgs. 3809-3819 (1992)).

In Huber et al., 1986, Arch. Microbiol. 144:324-333, the isolation ofthe bacterium Thermotoga maritima is described. T. maritima is aeubacterium that is strictly anaerobic, rod-shaped, fermentative,hyperthermophilic, and grows between 55° C. and 90° C., with an optimumgrowth temperature of about 80° C. This eubacterium has been isolatedfrom geothermally heated sea floors in Italy and the Azores. T. maritimacells have a sheath-like structure and monotrichous flagellation. T.maritima is classified in the eubacterium kingdom by virtue of havingmurein and fatty acid-containing lipids, diphtheria-toxin-resistantelongation factor 2, an RNA polymerase subunit pattern, and sensitivityto antibiotics.

The polynucleotide sequence and polypeptide encoded thereby of thepresent invention has been putatively identified as an endoglucanasehaving carboxymethyl cellulose activity.

In accordance with one aspect of the present invention, there isprovided a novel enzyme, as well as active fragments, analogs andderivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding an enzyme of thepresent invention including mRNAs, DNAs, cDNAs, genomic DNAs as well asactive analogs and fragments of such enzymes.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding anenzyme of the present invention, under conditions promoting expressionof said enzyme and subsequent recovery of said enzyme.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such enzyme, or polynucleotideencoding such enzyme for degradation of cellulose for the conversion ofplant biomass into fuels and chemicals, may also be used in detergents,the textile industry, in animal feed, in waste treatment, and in thefruit juice/brewing industry for the clarification and extraction ofjuices.

In accordance with yet a further aspect of the present invention, thereis also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to a nucleic acidsequence of the present invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such enzymes, or polynucleotidesencoding such enzymes, for in vitro purposes related to scientificresearch, for example, to generate probes for identifying similarsequences which might encode similar enzymes from other organisms byusing certain regions, i.e., conserved sequence regions, of thenucleotide sequence.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A-1C are an illustration of the full-length DNA and correspondingdeduced amino acid sequence of the enzyme of the present invention.Sequencing was performed using a 378 automated DNA sequencer (AppliedBiosystems, Inc.).

The present invention provides a purified thermostable enzyme thatcatalyzes the hydrolysis of the beta 1,4 glycosidic bonds in celluloseto thereby degrade cellulose. The purified enzyme is a carboxymethylcellulose from T. maritima which is a thermophilic eubacteria whichgrows in temperatures up to 90° C. The organism is strictly anaerobic,rod-shaped and fermentative, and grows between 55 and 90° C. (optimallyat 80° C.) . Thermotoga maritima is a representative of the genusThermotoga.

In a preferred embodiment, the CMCase enzyme of the present inventionhas a molecular weight of about 35 kilodaltons as measured by SDS-PAGEgel electrophoresis and an inferred molecular weight from the nucleotidesequence of the gene. This purified enzyme may be used to catalyze theenzymatic degradation of cellulose where desired.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singleMRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequencesultimately process to produce the desired protein.

“Recombinant” enzymes refer to enzymes produced by recombinant DNAtechniques; i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired enzyme. “Synthetic” enzymes are thoseprepared by chemical synthesis.

A DNA “coding sequence of” or a “nucleotide sequence encoding” aparticular enzyme, is a DNA sequence which is transcribed and translatedinto an enzyme when placed under the control of appropriate regulatorysequences. A “promotor sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. The promoter is part of theDNA sequence. This sequence region has a start codon at its 3′ terminus.The promoter sequence does include the minimum number of bases withelements necessary to initiate transcription at levels detectable abovebackground. However, after the RNA polymerase binds the sequence andtranscription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the matureenzyme having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) orfor the mature enzyme encoded by the cDNA contained in the pBluescriptII clone deposited as ATCC Deposit No. 97245.

This invention, in addition to the isolated nucleic acid moleculesencoding a CMCase enzyme, also provides substantially similar sequences.Isolated nucleic acid sequences are substantially similar if: (i) theyare capable of hybridizing under stringent conditions, hereinafterdescribed, to SEQ ID NO:1; (ii) or they encode DNA sequences which aredegenerate to SEQ ID NO:1. Degenerate DNA sequences encode the aminoacid sequence of SEQ ID NO:2, but have variations in the nucleotidecoding sequences. As used herein, substantially similar refers to thesequences having similar identity to the sequences of the instantinvention. The nucleotide sequences that are substantially the same canbe identified by hybridization or by sequence comparison. Enzymesequences that are substantially the same can be identified by one ormore of the following: proteolytic digestion, gel electrophoresis and/ormicrosequencing.

The polynucleotide of this invention was originally recovered from agenomic gene library derived from the organism Thermotoga maritima, ashereinafter described. It contains an open reading frame encoding aprotein of 317 amino acid residues. The protein exhibits the highestdegree of homology to endo-1, 4-beta-glucanase D from Clostridiumcellulolyticum with 36% identity at the amino acid level, and 17.2%identity at the DNA level.

One means for isolating a nucleic acid molecule encoding a CMCase enzymeis to probe a genomic gene library with a natural or artificiallydesigned probe using art recognized procedures (see, for example:Current Protocols in Molecular Biology, Ausubel F. M. et al. (EDS.)Green Publishing Company Assoc. and John Wiley Interscience, New York,1989, 1992). It is appreciated to one skilled in the art that SEQ IDNO:1, or fragments thereof (comprising at least 15 contiguousnucleotides), is a particularly useful probe. Other particular usefulprobes for this purpose are hybridizable fragments to the sequences ofSEQ ID NO:1 (i.e., comprising at least 15 contiguous nucleotides).

With respect to nucleic acid sequences which hybridize to specificnucleic acid sequences disclosed herein, hybridization may be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 0.5×SSC and 0.1% SDS at a temperature of 20 or 30° below the meltingtemperature of the probe, or even conditions represented by a washstringency of 0.1×SSC and 0.1% SDS at a temperature of 10° below themelting temperature of the DNA sequence to target DNA) in a standardhybridization assay. See J. Sambrook et al., Molecular Cloning, ALaboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory).

It is also appreciated that such probes can be and are preferablylabeled with an analytically detectable reagent to facilitateidentification of the probe. Useful reagents include but are not limitedto radioactivity, fluorescent dyes or enzymes capable of catalyzing theformation of a detectable product. The probes are thus useful to isolatecomplementary copies of DNA from other animal sources or to screen suchsources for related sequences.

The coding sequence for the CMCase enzyme of the present invention wasidentified by preparing a T. maritima genomic DNA library and screeningthe library for the clones having CMCase activity. Such methods forconstructing a genomic gene library are well-known in the art. One meanscomprises shearing DNA isolated from T. maritima by physical disruption.A small amount of the sheared DNA is checked on an agarose gel to verifythat the majority of the DNA is in the desired size range (approximately3-6 kb). The DNA is then blunt ended using Mung Bean Nuclease, incubatedat 37° C. and phenol/chloroform extracted. The DNA is then methylatedusing Eco RI Methylase. Eco RI linkers are then ligated to the bluntends through the use of T4 DNA ligase and incubation at 4° C. Theligation reaction is then terminated and the DNA is cut-back with Eco RIrestriction enzyme. The DNA is then size fractionated on a sucrosegradient following procedures known in the art, for example, Maniatis,T., et al., Molecular Cloning, Cold Spring Harbor Press, New York, 1982,which is hereby incorporated by reference in its entirety.

A plate assay is then performed to get an approximate concentration ofthe DNA. Ligation reactions are then performed and 1 μl of the ligationreaction is packaged to construct a library. The library is thenamplified.

The gene and gene products of the present invention may also be used asa probe to isolate other nucleic acid sequences and other enzymes uponisolation and expression, which may then be measured for retention ofbiological activity characteristic to the enzyme of the presentinvention, for example, in an assay for detecting enzymatic CMCaseactivity. Such enzymes include truncated forms of CMCase, and variantssuch as deletion and insertion variants.

An example of such an assay is an assay for the detection ofendogluconase activity based on specific interaction of direct dyes suchas Congo red with polysaccharides. This colorant reacts withbeta-1,4-glucans causing a visible red shift (Wood, P. J., Carbohydr.Res., 85:271 (1980) and Wood, P. J., Carbohydr. Res., 94:cl9 (1981)).The preferred substrate for the test is carboxymethylcellulose (CMC)which can be obtained from different sources (Hercules Inc., Wilmington,Del., Type 4M6F or Sigma Chemical Company, St. Louis, Mo., MediumViscosity). The CMC is incorporated as the main carbon source into aminimal agar medium in quantities of 0.1-1.0%. The microorganisms can bescreened directly on these plates, but the replica plating techniquefrom a master plate is preferable since the visualization of theactivity requires successive floodings with the reagents, which wouldrender the reisolation of active colonies difficult. Suchendoglucanase-producing colonies are detectable after a suitableincubation time (1-3 days depending on the growth), by flooding theplate with 10 ml of a 0.1% aqueous solution of Congo Red. The colorationis terminated after 20 minutes by pouring off the dye and flooding theplates with 10 ml of 5M NaCl solution (commercial salt can be used).After an additional 20 minutes, the salt solution is discarded andendoglucanase activity is revealed by a pale-orange zone around theactive microorganisms. In some cases, these zones can be enhanced bytreating the plates with 1 N acetic acid, causing the dye to change itscolor to blue.

The same technique can be used as a cup-plate diffusion assay withexcellent sensitivity for the determination of CMCase activity inculture filtrates or during enzyme purification steps (Carger, J. H.,Anal. Biochem., 153:75 (1986)). See generally, Methods for MeasuringCellulose Activities, Methods in Enzymology, Vol. 160, pgs. 87-116.

The enzyme of the present invention has enzymatic activity with respectto the hydrolysis of the beta 1,4 glycosidic bonds incarboxymethylcellulose, since the halos discussed above are shown aroundthe colonies.

The polynucleotide of the present invention may be in the form of DNAwhich DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may bedouble-stranded or single-stranded, and if single stranded may be thecoding strand or non-coding (anti-sense) strand. The coding sequencewhich encodes the mature enzyme may be identical to the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or that of the deposited clone or may be adifferent coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same matureenzyme as the DNA of FIG. 1 (SEQ ID NO:1) or the deposited DNA.

The polynucleotide which encodes for the mature enzyme of FIG. 1 (SEQ IDNO:2) or for the mature enzyme encoded by the deposited cDNA mayinclude, but is not limited to: only the coding sequence for the matureenzyme; the coding sequence for the mature enzyme and additional codingsequence such as a leader sequence or a proprotein sequence; the codingsequence for the mature enzyme (and optionally additional codingsequence) and non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature enzyme.

Thus, the term “polynucleotide encoding an enzyme (protein)” encompassesa polynucleotide which includes only coding sequence for the enzyme aswell as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the enzyme having the deduced amino acid sequence of FIG.1 (SEQ ID NO:2) or the enzyme encoded by the cDNA of the depositedclone. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature enzyme as shown in FIG. 1 (SEQ ID NO:2) or the same mature enzymeencoded by the cDNA of the deposited clone as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the enzyme of FIG. 1 (SEQ ID NO:2) or the enzyme encoded bythe cDNA of the deposited clone. Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded enzyme.

The present invention also includes polynucleotides, wherein the codingsequence for the mature enzyme may be fused in the same reading frame toa polynucleotide sequence which aids in expression and secretion of anenzyme from a host cell, for example, a leader sequence which functionsto control transport of an enzyme from the cell. The enzyme having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the enzyme. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA or a genomic library to isolate thefull length DNA and to isolate other DNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 10, preferably at least 15, and even morepreferably at least 30 bases and may contain, for example, at least 50or more bases. The probe may also be used to identify a DNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of genomic DNA to determine which members ofthe library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode enzymes which eitherretain substantially the same biological function or activity as themature enzyme encoded by the DNA of FIG. 1 (SEQ ID NO:1) or thedeposited DNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed asprobes forthe polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the enzyme of SEQID NO:2 as well as fragments thereof, which fragments have at least 30bases and preferably at least 50 bases and to enzymes encoded by suchpolynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the enzymes encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a enzyme which has the deducedamino acid sequence of FIG. 1 (SEQ ID NO:2) or which has the amino acidsequence encoded by the deposited DNA, as well as fragments, analogs andderivatives of such enzyme.

The terms “fragment,” “derivative” and “analog” when referring to theenzyme of FIG. 1 (SEQ ID NO:2) or that encoded by the deposited DNA,means a enzyme which retains essentially the same biological function oractivity as such enzyme. Thus, an analog includes a proprotein which canbe activated by cleavage of the proprotein portion to produce an activemature enzyme.

The enzyme of the present invention may be a recombinant enzyme, anatural enzyme or a synthetic enzyme, preferably a recombinant enzyme.

The fragment, derivative or analog of the enzyme of FIG. 1 (SEQ ID NO:2)or that encoded by the deposited DNA may be (i) one in which one or moreof the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature enzyme is fused with another compound, such as a compound toincrease the half-life of the enzyme (for example, polyethylene glycol),or (iv) one in which the additional amino acids are fused to the matureenzyme, such as a leader or secretory sequence or a sequence which isemployed for purification of the mature enzyme or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

The enzymes and polynucleotides of the present invention are preferablyprovided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

The enzymes of the present invention include the enzyme of SEQ ID NO:2(in particular the mature enzyme) as well as enzymes which have at least70% similarity (preferably at least 70% identity) to the enzyme of SEQID NO:2 and more preferably at least 90% similarity (more preferably atleast 90% identity) to the enzyme of SEQ ID NO:2 and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the enzyme of SEQ ID NO:2 and also include portions of suchenzymes with such portion of the enzyme generally containing at least 30amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two enzymes is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one enzyme to the sequence of a second enzyme.

Fragments or portions of the enzymes of the present invention may beemployed for producing the corresponding full-length enzyme by peptidesynthesis; therefore, the fragments may be employed as intermediates forproducing the full-length enzymes. Fragments or portions of thepolynucleotides of the present invention may be used to synthesizefull-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof enzymes of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing enzymes by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing an enzyme. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures ad others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pD10, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia);Eukaryotic: pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG,pSVL (Pharmacia). However, any other plasmid or vector may be used aslong as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the enzymes of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the enzymes of the present inventionby higher eukaryotes is increased by inserting an enhancer sequence intothe vector. Enhancers are cis-acting elements of DNA, usually about from10 to 300 bp that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, a cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated enzyme. Optionally, the heterologoussequence can encode a fusion enzyme including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsite, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The enzyme can be recovered and purified from recombinant cell culturesby methods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The enzymes of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the enzymes of the present invention may beglycosylated or may be non-glycosylated. Enzymes of the invention may ormay not also include an initial methionine amino acid residue.

The enzyme of this invention may be employed for any purpose in whichsuch enzyme activity is necessary or desired. In a preferred embodimentthe enzyme is employed for catalyzing the hydrolysis of cellulose. Thedegradation of cellulose may be used for the conversion of plant biomassinto fuels and chemicals.

The enzyme of the present invention may also be employed in detergents,the textile industry, in animal feed, in waste treatment and in thefruit juice/brewing industry for the clarification and extraction ofjuices.

In a preferred embodiment, the enzyme of the present invention is athermostable enzyme which is stable to heat and is heat resistant andcatalyzes the enzymatic hydrolysis of cellulose, i,e., the enzyme isable to renature and regain activity after brief (i.e., 5 to 30 seconds)exposure to temperatures of 80° C. to 105° C. and has a temperatureoptimum of above 60° C.

The enzymes, their fragments or other derivatives, or analogs thereof,or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the enzymes corresponding to a sequence ofthe present invention can be obtained by direct injection of the enzymesinto an animal or by administering the enzymes to an animal, preferablya nonhuman. The antibody so obtained will then bind the enzymes itself.In this manner, even a sequence encoding only a fragment of the enzymescan be used to generate antibodies binding the whole native enzymes.Such antibodies can then be used to isolate the enzyme from cellsexpressing that enzyme.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic enzyme products of this invention. Also, transgenic mice maybe used to express humanized antibodies to immunogenic enzyme productsof this invention.

Antibodies generated against the enzyme of the present invention may beused in screening for similar enzymes from other organisms and samples.Such screening techniques are known in the art, for example, one suchscreening assay is described in “Methods for Measuring CelluloseActivities”, Methods in Enzymology Vol 160, pp. 87-116, which is herebyincorporated by reference in its entirety.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of CMCase

A T. maritima genomic library was constructed in the Lambda ZapII®cloning vector (Stratagene Cloning Systems), and mass excision wasperformed according to the manufacturers protocol to yield a genelibrary in the pBluescript cloning vector. The pBluescript library wasscreened in SOLR E. Coli cells (Stratagene) for CMCase activity and apositive clone was identified and isolated. This clone was used toinoculate an overnight culture of Luria Broth liquid medium as perAusubel, F. M., et al., Short Protocols in Molecular Biology, 2d Ed.,Harvard Medical School (1992). The plasmid DNA was isolated from theovernight culture using an alkaline lysis mini-prep protocol as perManiatis, T., et al., Molecular Cloning, Cold Spring Harbor Press, NewYork (1982). Mini-prep DNA was then used to transform competent E. colicells, XL1 blue (Stratagene) according to the manufacturer's protocol. Asingle clone was then used to innoculate a 100 ml overnight culture ofLuria Broth liquid medium and plasmid DNA was isolated from thisovernight using midi-prep procedure according to the manufacturer'sprotocol (Qiagen). The midi-prep plasmid DNA was partially sequencedwith an ABI 377 and a putative open reading frame was idnetified withinthe sequenced region. The sequence information was used in thegeneration of primer sequences which were subsequently used to PCRamplify the target gene encoding the CMCase activity. The primersequences used were as follows:

5′ TTATTGCGGCCGCTTAAGGAGGAAAAATTATGGGTGTTGATCCTTTTGAA 3′ (SEQ. ID NO: 3)and

5′ TTATTGGATCCGAAGGTTGAAACCACGCCATCT 3′ (SEQ. ID NO: 4).

The amplification product was subcloned into the pBluescript II cloningvector (Stratagene). The plasmid clone was transformed in to XL1 Bluecells again for verification. The plasmid clone contains the DNAencoding the CMCase enzyme of the present invention as shown in FIG. 1and deposited as ATCC No. 97245.

A pBLuescript II clone containing the DNA encoding the enzyme of thepresent invention may be obtained from the ATCC, ATCC Deposit No.97245.This pBluescript II clone containing the DNA of the present invention isused to transform E. coli XL1 Blue cells and the E. coli XL1 Blue cellsare used to inoculate a 5 ml overnight culture of Luria Broth liquidmedium. The 5 ml culture was aliquoted into 1 ml aliquots, and eachaliquot was used to innoculate 1 liter of 5X LB culture media. Cellswere grown overnight in five 2-liter shake flasks at 37° C. Each oneliter cell culture pellet was resuspended in 150 ml of 25 mM Tris, pH8.0 and then spun at 4K rpm for 10 minutes at 4° C. The resulting pelletwas resuspended in 5 ml of 25 mM Tris, pH 8.0, and sonicated with amicrosonicator tip 10 times at 30 second intervals. The cell debris wasspun out in a SS-34 rotor at 12K rpms for 10 minutes at 4° C. Theresulting supernatant was then brought up to 10% ethanol and incubatedat 75° C. for 20 minutes. The flocculated proteins were spun out in anSS-34 rotor at 10K rpms for 10 minutes at 4° C. The resultantsupernatant was then filtered through a 0.22 micron filter and appliedto a weak anion exchange column (Poros, PI). The column was eluted witha 250, 500, 800 mM NaCl step in a 10 mM Tris Base/10 mM Bis Tris Propanebuffer at pH 8.0 (anion buffer). The active CMCase fraction came off atthe 250 mM step. This fraction was then diluted with the anion buffer toa concentration of 50 mM. It was then applied to a strong anion exchangecolumn (Poros, HQ) and the column was eluted with a 10 column volumegradient from 50 to 250 mM NaCl using anion buffer. A one band fractionof a 35 kD cellulose comes off in this gradient at approximately 150 mMNaCl.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described. On Aug. 29,1995, a deposit ofbiologically samples identified above were made with the American TypeCultute Collection (ATCC), having an address at 10801 UniversityBoulevard, Manassa, Va. 20110-2209, U.S.A. The deposits were made underthe provisions of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure andthe Regulations thereunder (Budapest Treaty). This assures maintenanceof viable cultures for a period of thirty (30) years from the date ofdeposit and at least five (5) years after the most recent request forthe furnishing of a sample of the deposit by the depository. Theorganisms will be made available by the ATCC under the terms of theBudapest Treaty, which assures permanent and unrestricted availabilityof the cultures to one determined by the U.S. Commissioner of Patentsand Trademarks to be entitled thereto according to 35 USC §122 and theCommissioner's rules pursuant thereto (including 37 CFR §1.12).

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35USC §112. A license may be required to make, use, or sell the depositedmaterials, and no such license is hereby granted.

4 954 BASE PAIRS NUCLEIC ACID SINGLE LINEAR cDNA unknown 1 ATGGGTGTTGATCCTTTTGA AAGGAACAAA ATATTGGGAA GAGGCATTAA TATAGGAAAT 60 GCGCTTGAAGCACCAAATGA GGGAGACTGG GGAGTGGTGA TAAAAGATGA GTTCTTCGAC 120 ATTATAAAAGAAGCCGGTTT CTCTCATGTT CGAATTCCAA TAAGATGGAG TACGCACGCT 180 TACGCGTTTCCTCCTTATAA AATCATGGAT CGCTTCTTCA AAAGAGTGGA TGAAGTGATA 240 AACGGAGCCCTGAAAAGAGG ACTGGCTGTT GCTATAAATA TTCATCACTA CGAGGAGTTA 300 ATGAATGATCCAGAAGAACA CAAGGAAAGA TTTCTTGCTC TTTGGAAACA AATTGCTGAT 360 CGTTATAAAGACTATCCCGA AACTCTATTT TTTGAAATTC TGAATGAACC TCACGGAAAT 420 CTTACTCCGGAAAAATGGAA TGAACTGCTT GAGGAAGCTC TAAAAGTTAT AAGATCAATT 480 GACAAAAAGCACACTATAAT TATAGGCACA GCTGAATGGG GGGGTATATC TGCCCTTGAA 540 AAACTGTCTGTCCCAAAATG GGAAAAAAAT TCTATAGTTA CAATTCACTA CTACAATCCT 600 TTCGAATTTACCCATCAAGG AGCTGAGTGG GTGGAAGGAT CTGAGAAATG GTTGGGAAGA 660 AAGTGGGGATCTCCAGATGA TCAGAAACAT TTGATAGAAG AATTCAATTT TATAGAAGAA 720 TGGTCAAAAAAGAACAAAAG ACCAATTTAC ATAGGTGAGT TTGGTGCCTA CAGAAAAGCT 780 GACCTTGAATCAAGAATAAA ATGGACCTCC TTTGTCGTTC GCGAAATGGA GAAAAGGAGA 840 TGGAGCTGGGCATACTGGGA ATTTTGTTCC GGTTTTGGTG TTTATGATAC TCTGAGAAAA 900 ACCTGGAATAAAGATCTTTT AGAAGCTTTA ATAGGAGGAG ATAGCATTGA ATAA 954 317 AMINO ACIDSAMINO ACID LINEAR PROTEIN unknown 2 Met Gly Val Asp Pro Phe Glu Arg AsnLys Ile Leu Gly Arg Gly Ile 5 10 15 Asn Ile Gly Asn Ala Leu Glu Ala ProAsn Glu Gly Asp Trp Gly Val 20 25 30 Val Ile Lys Asp Glu Phe Phe Asp IleIle Lys Glu Ala Gly Phe Ser 35 40 45 His Val Arg Ile Pro Ile Arg Trp SerThr His Ala Tyr Ala Phe Pro 50 55 60 Pro Tyr Lys Ile Met Asp Arg Phe PheLys Arg Val Asp Glu Val Ile 65 70 75 80 Asn Gly Ala Leu Lys Arg Gly LeuAla Val Ala Ile Asn Ile His His 85 90 95 Tyr Glu Glu Leu Met Asn Asp ProGlu Glu His Lys Glu Arg Phe Leu 100 105 110 Ala Leu Trp Lys Gln Ile AlaAsp Arg Tyr Lys Asp Tyr Pro Glu Thr 115 120 125 Leu Phe Phe Glu Ile LeuAsn Glu Pro His Gly Asn Leu Thr Pro Glu 130 135 140 Lys Trp Asn Glu LeuLeu Glu Glu Ala Leu Lys Val Ile Arg Ser Ile 145 150 155 160 Asp Lys LysHis Thr Ile Ile Ile Gly Thr Ala Glu Trp Gly Gly Ile 165 170 175 Ser AlaLeu Glu Lys Leu Ser Val Pro Lys Trp Glu Lys Asn Ser Ile 180 185 190 ValThr Ile His Tyr Tyr Asn Pro Phe Glu Phe Thr His Gln Gly Ala 195 200 205Glu Trp Val Glu Gly Ser Glu Lys Trp Leu Gly Arg Lys Trp Gly Ser 210 215220 Pro Asp Asp Gln Lys His Leu Ile Glu Glu Phe Asn Phe Ile Glu Glu 225230 235 240 Trp Ser Lys Lys Asn Lys Arg Pro Ile Tyr Ile Gly Glu Phe GlyAla 245 250 255 Tyr Arg Lys Ala Asp Leu Glu Ser Arg Ile Lys Trp Thr SerPhe Val 260 265 270 Val Arg Glu Met Glu Lys Arg Arg Trp Ser Trp Ala TyrTrp Glu Phe 275 280 285 Cys Ser Gly Phe Gly Val Tyr Asp Thr Leu Arg LysThr Trp Asn Lys 290 295 300 Asp Leu Leu Glu Ala Leu Ile Gly Gly Asp SerIle Glu 305 310 315 51 BASE PAIRS NUCLEIC ACID SINGLE LINEAROligonucleotide unknown 3 TTATTGCGGC CGCTTAAGGA GGAAAAAATT ATGGGTGTTGATCCTTTTGA A 51 33 BASE PAIRS NUCLEIC ACID SINGLE LINEAR Oligonucleotideunknown 4 TTATTGGATC CGAAGGTTGA AACCACGCCA TCT 33

What is claimed is:
 1. An isolated polynucleotide selected from thegroup consisting of: (a) a polynucleotide encoding an enzyme havingcarboxymethyl cellulose activity and an amino acid sequence as get forthin SEQ ID NO:2, from amino acids 1 to 317; and (b) a polynucleotidewhich hybridizes to and which is at least 70% identical to apolynucleotide of (a) and which encodes an enzyme having carboxymethylcellulose activity and having a sequence as set forth in SEQ ID NO:2. 2.The polynucleotide of claim 1 wherein the polynucleotide is DNA.
 3. Thepolynucleotide of claim 1, wherein the polynucleotide has a sequence asset forth in SEQ ID NO:1 from nucleotide 1 to nucleotide
 951. 4. Anisolated polynucleotide which encodes an enzyme having the amino acidsequence as set forth in SEQ ID NO:2 from amino acids 1 to 317 andhaving ATCC Deposit No.
 97245. 5. A vector comprising the DNA of claim2.
 6. A host cell transformed with the vector of claim
 5. 7. A processfor producing a carboxymethyl cellulose comprising expressing from thehost cell of claim 6 the enzyme encoded by said DNA.
 8. A process forproducing cells capable of expressing a carboxymethyl cellulosecomprising transforming host cells with the vector of claim
 5. 9. Anisolated polynucleotide encoding a mature carboxymethyl cellulose havinga sequence as set forth in SEQ ID NO:2 from amino acid 1 to amino acid317.
 10. The polynucleotide of claim 9, wherein said polynucleotide isthe DNA of SEQ ID NO:1 from nucleotide 1 to nucleotide
 951. 11. Anisolated polynucleotide selected from the group consisting of: (a) SEQID NO:1, from nucleotide 1 to nucleotide 951; (b) SEQ ID NO:1, fromnucleotide 1 to nucleotide 951, wherein T can also be U; (c) nucleicacid sequences complementary to (a) and (b); and (d) fragments of (a),(b), or (c) that are at least 15 bases in length and that hybridize toDNA which encodes the amino acid sequences of SEQ ID NO:2, from aminoacid 1 to amino acid 317, under moderate to highly stringent conditions.